This proposal is to examine molecular mechanisms of HIV-mediated neuroinflammation in the presence of ART and opiods. We will use morphine as it exacerbates inflammation and CNS disease in many HIV infected people. HIV infection of the CNS results in chronic inflammation that leads to cognitive deficits in > 50% of infected people. This inflammation and subsequent CNS damage is not mitigated with ART. Inflammation is a key process in HIV disease and therapies to limit this and ongoing CNS viral seeding must be developed to improve the quality of life of infected people. This is even more pressing as HIV positive people live longer. HIV enters the CNS soon after peripheral infection and despite ART, persists within infected cells. HIV entry into the brain is mediated, at least in part, by infected monocyte transmigration across the blood brain barrier (BBB). Mature monocytes expressing CD14 and CD16 are key mediators of HIV neuropathogenesis. These monocytes are productively infected with HIV and primed to cross the BBB. Once within the CNS, they may differentiate into infected macrophages that can persist for years. This leads to infection and/or activation of CNS cells, including macrophages and microglia, resulting in chronic inflammation characterized by production of virus and/or viral proteins, and cytokines, and chemokines. Chemokines, in particular CCL2, increase transmigration of peripheral blood infected/uninfected monocytes, continuing inflammation and viral seeding of the CNS that mediates neuronal dendritic pruning and degeneration in a large number of infected people by mechanisms not well understood. ART does not eliminate cells harboring HIV. Thus, monocyte/macrophage activation, and production of HIV early proteins continue, resulting in brain injury despite successful ART. We will characterize effects of morphine, HIV, and ART on mechanisms that mediate monocyte entry into the CNS and on subsequent viral reseeding and neuroinflammation. We will use state of the art in vitro techniques, the powerful approach of single cell RNA sequencing, and transgenic mice to characterize potential therapeutics to limit inflammation and guide efficacy of ART. We will characterize the impact of morphine and ART on transmigration of HIV infected and uninfected human monocytes across a model of the human BBB and use scRNA-seq to identify unique genes expressed by individual transmigrating HIV-infected and HIV-exposed monocytes in the presence or absence of morphine; characterize the impact of HIV, ART, and/or morphine on the function of human macrophages and, using scRNA-seq, on expression of inflammatory genes by individual human macrophages; apply an HIV transgenic mouse model to evaluate the in vivo impact of opioids and HIV on inflammatory genes expressed in vivo by individual monocytes from the mice that transmigrated across the BBB, and by resident individual brain macrophages/microglia from these mice; and compare expression of inflammatory genes by individual macrophages/microglia isolated from the brains of HIV-nave and HIV- infected individuals including those from people who were on palliative opioid treatment using scRNA-seq.